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• W2098998632 abstract "Activity-based probes (ABPs) that specifically target subsets of related enzymatic proteins are finding increasing use in proteomics research. One of the main applications for these reagents is affinity isolation of probe-labeled targets. However, the use of cheap and efficient biotin affinity tags on ABPs can be problematic due to difficulty in release of captured proteins. Here we describe the evaluation of activity-based probes carrying a chemically cleavable linker that allows selective release of probe-labeled proteins under mild elution conditions that are compatible with mass spectrometric analysis. Specifically, we compare results from standard on-bead digestion of probe-labeled targets after affinity purification with the results obtained using chemoselective cleavage. Results are presented for multiple APBs that target both serine and cysteine proteases. These results highlight significant improvements in the quality of data obtained by using the cleavable linker system. Activity-based probes (ABPs) that specifically target subsets of related enzymatic proteins are finding increasing use in proteomics research. One of the main applications for these reagents is affinity isolation of probe-labeled targets. However, the use of cheap and efficient biotin affinity tags on ABPs can be problematic due to difficulty in release of captured proteins. Here we describe the evaluation of activity-based probes carrying a chemically cleavable linker that allows selective release of probe-labeled proteins under mild elution conditions that are compatible with mass spectrometric analysis. Specifically, we compare results from standard on-bead digestion of probe-labeled targets after affinity purification with the results obtained using chemoselective cleavage. Results are presented for multiple APBs that target both serine and cysteine proteases. These results highlight significant improvements in the quality of data obtained by using the cleavable linker system. Over the last decade, activity-based proteomics has become a prominent method for profiling enzyme activities in complex protein samples (for reviews, see Refs. 1Berger A.B. Vitorino P.M. Bogyo M. Activity-based protein profiling: applications to biomarker discovery, in vivo imaging and drug discovery.Am. J. Pharmacogenomics. 2004; 4: 371-381Crossref PubMed Scopus (101) Google Scholar, 2Evans M.J. Cravatt B.F. Mechanism-based profiling of enzyme families.Chem. Rev. 2006; 106: 3279-3301Crossref PubMed Scopus (458) Google Scholar, 3Fonović M. Bogyo M. Activity based probes for proteases: applications to biomarker discovery, molecular imaging and drug screening.Curr. Pharm. Des. 2007; 13: 253-261Crossref PubMed Scopus (109) Google Scholar, 4Jeffery D.A. Bogyo M. Chemical proteomics and its application to drug discovery.Curr. Opin. Biotechnol. 2003; 14: 87-95Crossref PubMed Scopus (204) Google Scholar, 5Sadaghiani A.M. Verhelst S.H. Bogyo M. Tagging and detection strategies for activity-based proteomics.Curr. Opin. Chem. Biol. 2007; 11: 20-28Crossref PubMed Scopus (185) Google Scholar). This approach is dependent on small molecules termed activity-based probes (ABPs) 1The abbreviations used are: ABP, activity-based probe; DIEA, N,N-diisopropylethylamine; PyBOP, benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate; Fmoc, N-(9-fluorenyl)methoxycarbonyl; DMF, N,N-dimethylformamide; UCH-L1, ubiquitin carboxyl-terminal hydrolase L1. 1The abbreviations used are: ABP, activity-based probe; DIEA, N,N-diisopropylethylamine; PyBOP, benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate; Fmoc, N-(9-fluorenyl)methoxycarbonyl; DMF, N,N-dimethylformamide; UCH-L1, ubiquitin carboxyl-terminal hydrolase L1. that use chemically reactive functional groups to covalently modify the active sites of a specific enzyme class. Activity-based probes have been specifically designed to target enzyme families with well established catalytic mechanisms including proteases, kinases, lipases, glycosylases, histone deacylases, and phosphatases (for a general review, see Ref. 2Evans M.J. Cravatt B.F. Mechanism-based profiling of enzyme families.Chem. Rev. 2006; 106: 3279-3301Crossref PubMed Scopus (458) Google Scholar). Among proteases, activity-based probes have been mainly used for profiling the serine and cysteine families because both utilize catalytic nucleophiles in their active site that can be effectively targeted by a number of classes of synthetically accessible electrophiles (6Bogyo M. Verhelst S. Bellingard-Dubouchaud V. Toba S. Greenbaum D. Selective targeting of lysosomal cysteine proteases with radiolabeled electrophilic substrate analogs.Chem. Biol. 2000; 7: 27-38Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 7Borodovsky A. Ovaa H. Kolli N. Gan-Erdene T. Wilkinson K.D. Ploegh H.L. Kessler B.M. Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family.Chem. Biol. 2002; 9: 1149-1159Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar, 8Greenbaum D. Medzihradszky K.F. Burlingame A. Bogyo M. Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools.Chem. Biol. 2000; 7: 569-581Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar, 9Kato D. Boatright K.M. Berger A.B. Nazif T. Blum G. Ryan C. Chehade K.A. Salvesen G.S. Bogyo M. Activity-based probes that target diverse cysteine protease families.Nat. Chem. Biol. 2005; 1: 33-38Crossref PubMed Scopus (285) Google Scholar, 10Liu Y. Patricelli M.P. Cravatt B.F. Activity-based protein profiling: the serine hydrolases.Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14694-14699Crossref PubMed Scopus (815) Google Scholar, 11Mahrus S. Craik C.S. Selective chemical functional probes of granzymes A and B reveal granzyme B is a major effector of natural killer cell-mediated lysis of target cells.Chem. Biol. 2005; 12: 567-577Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 12Pan Z. Jeffery D.A. Chehade K. Beltman J. Clark J.M. Grothaus P. Bogyo M. Baruch A. Development of activity-based probes for trypsin-family serine proteases.Bioorg. Med. Chem. Lett. 2006; 16: 2882-2885Crossref PubMed Scopus (60) Google Scholar).One strength of ABPs is that they allow direct monitoring of the regulation of enzyme activity, thereby providing information that can be used to infer the function of targets. Perhaps even more importantly, ABPs provide an efficient way to enrich complex proteomes thereby making subsequent analytical analysis much simpler. Biotin is often used as a tag to facilitate isolation of labeled proteins by affinity chromatography on immobilized avidin. Biotin is commonly used because it is relatively cheap and provides diffusion-limited binding to avidin, thus allowing even low abundance proteins to be isolated with high efficiency. The disadvantage of this high affinity interaction with avidin is the harsh denaturing conditions required to disrupt the biotin-avidin interaction. Because these conditions are not directly compatible with mass spectrometry applications, samples must typically first be analyzed by SDS-PAGE, and subsequently proteins must be cut from a gel for identification. This additional step leads to significant loss of sample and can cause problems for proteins that do not resolve well via gel electrophoresis. Alternatively, proteins can be directly digested from the avidin resin; however, contamination by avidin and endogenously biotinylated proteins can be significant and in many cases will prevent detection of low abundance targets. Furthermore the on-bead digestion method leads to significantly more false positives as a result of nonspecific binding of proteins to the avidin resin. These false positive are particularly problematic because hit validation is often the most costly and time-consuming phase of proteomics studies.To address some of the limitations of biotin, various cleavable linkers have been reported. These include acid-cleavable linkers (13van der Veken P. Dirksen E.H. Ruijter E. Elgersma R.C. Heck A.J. Rijkers D.T. Slijper M. Liskamp R.M. Development of a novel chemical probe for the selective enrichment of phosphorylated serine- and threonine-containing peptides.Chembiochem. 2005; 6: 2271-2280Crossref PubMed Scopus (61) Google Scholar), peptide linkers that can be cleaved by a proteolytic enzyme (14Speers A.E. Cravatt B.F. A tandem orthogonal proteolysis strategy for high-content chemical proteomics.J. Am. Chem. Soc. 2005; 127: 10018-10019Crossref PubMed Scopus (150) Google Scholar), and disulfide linkers (15Shimkus M. Levy J. Herman T. A chemically cleavable biotinylated nucleotide: usefulness in the recovery of protein-DNA complexes from avidin affinity columns.Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2593-2597Crossref PubMed Scopus (104) Google Scholar). Although all of these regents have proved advantageous for specific applications, all have disadvantages that limit their general use. In particular, elution by acidic cleavage is often nonspecific, and TFA must be removed from the sample prior to MS analysis. Furthermore proteolytic elution requires introduction of an additional protease and tends to be less practical because elution efficiency is strongly dependent on protease activity. Probes that utilize a disulfide-cleavable linker are not compatible with buffers containing reducing reagents such as DTT and are prematurely cleaved under many cellular conditions.We have recently developed a diazobenzene linker that can be cleaved to the corresponding anilines using a mild reducing agent (16Verhelst S.H. Fonović M. Bogyo M. A mild chemically cleavable linker system for functional proteomic applications.Angew. Chem. Int. Ed. Engl. 2007; 46: 1284-1286Crossref PubMed Scopus (122) Google Scholar). We present here the analysis of this cleavable linker system applied to ABPs that target both cysteine and serine proteases. When compared with the on-bead digestion of labeled targets, specific chemical elution produces more reliable proteomics data that are free of background protein contamination.RESULTS AND DISCUSSIONWe have recently reported the synthesis of the probe SV1 that specifically targets papain family cysteine proteases (16Verhelst S.H. Fonović M. Bogyo M. A mild chemically cleavable linker system for functional proteomic applications.Angew. Chem. Int. Ed. Engl. 2007; 46: 1284-1286Crossref PubMed Scopus (122) Google Scholar). This probe has an epoxide warhead and a biotin tag and contains the diazobenzene cleavable linker that can be specifically cleaved with greater than 90% elution efficiency using mild reducing conditions (16Verhelst S.H. Fonović M. Bogyo M. A mild chemically cleavable linker system for functional proteomic applications.Angew. Chem. Int. Ed. Engl. 2007; 46: 1284-1286Crossref PubMed Scopus (122) Google Scholar). Using this cleavable ABP, labeled target cysteine proteases are affinity-purified using an avidin resin and then eluted by cleavage of the diazobenzene linker using sodium hydrosulfite (Fig. 1). We therefore decided to use this general probe as a starting point to evaluate the utility of the cleavable linker system for activity-based protein profiling applications.When using standard (non-cleavable) activity-based probes labeled with biotin, enriched proteins must be prepared for MS analysis in either of two ways. They can be eluted by denaturation by boiling in the presence of SDS, analyzed by SDS-PAGE, and digested “in gel,” or they can be digested on bead. In-gel digestion is time-consuming, requires higher sample quantity, and is more prone to keratin contamination. On-bead digestion is one of the most commonly used non-gel methods for proteomics analysis of affinity-purified targets (19Fu Z. Larson K.A. Chitta R.K. Parker S.A. Turk B.E. Lawrence M.W. Kaldis P. Galaktionov K. Cohn S.M. Shabanowitz J. Hunt D.F. Sturgill T.W. Identification of yin-yang regulators and a phosphorylation consensus for male germ cell-associated kinase (MAK)-related kinase.Mol. Cell. Biol. 2006; 26: 8639-8654Crossref PubMed Scopus (59) Google Scholar, 20Jessani N. Niessen S. Wei B.Q. Nicolau M. Humphrey M. Ji Y. Han W. Noh D.Y. Yates III, J.R. Jeffrey S.S. Cravatt B.F. A streamlined platform for high-content functional proteomics of primary human specimens.Nat. Methods. 2005; 2: 691-697Crossref PubMed Scopus (201) Google Scholar, 21Labugger R. Simpson J.A. Quick M. Brown H.A. Collier C.E. Neverova I. Van Eyk J.E. Strategy for analysis of cardiac troponins in biological samples with a combination of affinity chromatography and mass spectrometry.Clin. Chem. 2003; 49: 873-879Crossref PubMed Scopus (46) Google Scholar, 22Tagwerker C. Flick K. Cui M. Guerrero C. Dou Y. Auer B. Baldi P. Huang L. Kaiser P. A tandem affinity tag for two-step purification under fully denaturing conditions: application in ubiquitin profiling and protein complex identification combined with in vivo cross-linking.Mol. Cell. Proteomics. 2006; 5: 737-748Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). In this approach, enriched proteins are denatured, reduced, alkylated, and digested while they are bound to affinity matrix such as immobilized avidin. However, natively biotinylated and abundant nonspecifically bound proteins can still be present during the digestion step even after stringent washing. These proteins produce false positives and can interfere with identification of low abundance targets.One of the most significant advantages of the cleavable linker strategy is that it avoids background resulting from nonspecifically retained or endogenously biotinylated proteins. However, for the cleavable linker to be of real value it must allow highly efficient elution of targets. To test the sensitivity of the cleavable probe we directly compared samples of labeled proteins isolated by on-bead digestion and by chemical cleavage (Fig. 2). We labeled mouse liver, kidney, and spleen with the SV1 probe and divided each sample into two aliquots. One was digested on bead, whereas the other proteins were chemically eluted from the beads by linker cleavage and digested in solution as described under “Experimental Procedures.” In addition, a small amount of each proteome was labeled with the related fluorescent probe SV31 (Fig. 2A). This probe allows direct analysis of labeled proteomes by SDS-PAGE followed by scanning of the gel with a laser scanner. As expected the labeling profiles were similar in all three proteomes, and the pattern of labeled proteins that were blocked by pretreatment with the general inhibitor E-64 corresponded to the patterns observed previously for labeling of cathepsins B, X, H, and C by the related probe DCG-04 (23Greenbaum D. Baruch A. Hayrapetian L. Darula Z. Burlingame A. Medzihradszky K.F. Bogyo M. Chemical approaches for functionally probing the proteome.Mol. Cell. Proteomics. 2002; 1: 60-68Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). Consistent with these reported patterns of labeling, cathepsin X (also called cathepsin Z) was resolved above cathepsins B and H on the gel even though its molecular weight based on sequence alone suggests it should be smaller than both cathepsins B and H. This may be due to differences in secondary structure or post-translational modifications, or effects based on detergent denaturation. In all three proteomes on-bead digestion yielded samples contaminated with background proteins. The main contaminant was propionyl-coenzyme A carboxylase, which is a natively biotinylated protein. Other identified background contaminants were abundant tissue proteins such as hemoglobin, albumin, and transferases (Fig. 2B). The highest contamination was observed in liver and kidney proteomes, where over 60% of all identified peptides corresponded to background proteins. Contamination was not as high in spleen lysate due to the absence of carboxylases in this tissue. Samples obtained from chemical cleavage on the other hand showed a minimal number of background proteins while retaining good coverage of previously identified cysteine proteases (23Greenbaum D. Baruch A. Hayrapetian L. Darula Z. Burlingame A. Medzihradszky K.F. Bogyo M. Chemical approaches for functionally probing the proteome.Mol. Cell. Proteomics. 2002; 1: 60-68Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). Importantly the overall sequence coverage of the identified cathepsin targets was virtually identical for both methods suggesting that elution is nearly quantitative as reported previously for this probe (16Verhelst S.H. Fonović M. Bogyo M. A mild chemically cleavable linker system for functional proteomic applications.Angew. Chem. Int. Ed. Engl. 2007; 46: 1284-1286Crossref PubMed Scopus (122) Google Scholar). As a further indication of the selectivity of the linker system, on-bead digestion of the kidney proteome failed to identify one of the primary cathepsin target proteins (cathepsin C), whereas chemical cleavage provided confident identification of cathepsin C with 18% sequence coverage. Taken together these results suggest that chemical cleavage of the diazobenzene linker is highly efficient and results in significant reduction in background signals, thus reducing overall false negatives produced by abundant background proteins.Fig. 2Comparison of target identification by on-bead digestion and chemical elution.A, structure of the cleavable fluorescent probe SV31 that allows direct analysis of probe-labeled proteins by SDS-PAGE followed by scanning with a laser scanner. B, mouse liver, kidney, and spleen lysates were labeled with SV31 for SDS-PAGE analysis (left panels) and by the SV1 probe for direct mass spectrometric analysis (right panels). Fluorescent protein bands that are competed by pretreatment with the cysteine protease inhibitor E-64 were identified as cathepsins. After enrichment on immobilized avidin, each of the three labeled proteomes was divided into two aliquots. One was prepared for LC-MS/MS analysis by on-bead digestion, whereas the other was chemically eluted and digested in solution. Proteins identified in each tissue lysate are listed with their accession number and theoretical molecular mass. To compare both approaches, the number of identified peptides and percentage of amino acid sequence coverage are listed for each protein. Please note that molecular masses of cathepsins listed in the tables are for unprocessed zymogens and are therefore significantly larger than the masses of processed, active cathepsins observed on the SDS-PAGE gels. cat, cathepsin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We also applied the cleavable probe SV1 to brain lysate to determine whether the reduced background and high sensitivity observed in liver, spleen, and kidney samples could be observed in other tissues that have typically produced high background in our activity-based protein profiling experiments (Fig. 3). SDS-PAGE analysis of brain lysate incubated with the fluorescent probe SV31 showed labeling of two cysteine protease activities that could be inhibited by E-64 (Fig. 3A). Based on their molecular weight and inhibitor sensitivity, these two proteases were predicted to be cathepsins X and B. Direct on-bead digestion of enriched brain lysate identified a total 11 proteins, but only two of them were positively identified as cysteine proteases (cathepsin B and ubiquitin carboxyl-terminal hydrolase L1; Fig. 3B). In contrast, MS analysis of the chemically cleaved sample identified four proteins, and two of these were identified as cysteine proteases (cathepsins B and X; Fig 3B). As cathepsin B, cathepsin X, and ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) are all members of the papain family of cysteine proteases, they all represent potential targets of the SV1 probe. However, UCH-L1 is a highly abundant protein that makes up over 1% of total brain protein content (24Wilkinson K.D. Lee K.M. Deshpande S. Duerksen-Hughes P. Boss J.M. Pohl J. The neuron-specific protein PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase.Science. 1989; 246: 670-673Crossref PubMed Scopus (755) Google Scholar) making it a potential nonspecific background protein. Because SDS-PAGE did not show any fluorescently labeled band at the predicted size of UCH-L1 and the SV1 probe also failed to label recombinant UCH-L1, 2M. Fonović, unpublished results. we concluded that UCH-L1 is in fact a false positive background protein that was only identified in the on-bead sample. Furthermore the weakly labeled 32-kDa protein that can be blocked by E-64 pretreatment (Fig. 3A) was identified as cathepsin X in the chemical cleavage sample but was missed in the on-bead digestion sample. Thus, the chemical cleavage method both prevented identification of a false positive protein that seemed to be a legitimate target and helped to identify a false negative target that was missed by on-bead digestion due to high background contamination. In addition, chemical cleavage produced clean MS/MS spectra that allowed confident identification of probe-labeled cathepsin X (Fig. 3C), suggesting that the enhanced selectivity does not come at the cost of reduced spectral quality in the raw MS data.Fig. 3Determination of SV1 probe targets in mouse brain lysate. Mouse brain lysate was labeled with SV31 for SDS-PAGE analysis (A) and with SV1 for direct mass spectrometric analysis (B). Targets were enriched by affinity binding to immobilized avidin and eluted by chemical cleavage or digested on beads. All identified proteins determined with chemical cleavage and on-bead digestion are listed with their number of identified peptides and amino acid sequence coverage (B). C, MS/MS spectra for both cathepsin X peptides determined in the sample prepared by chemical elution are shown together with their charge and Sequest XCorr and ΔCn values. Cat, cathepsin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To show applicability of the cleavable linker approach for profiling of other catalytic classes of proteases in the context of other probe scaffolds, we incorporated the diazobenzene linker into the peptide backbone of the commercially available probe DAP22C to make the probe MTS-I-49 (Enzyme Systems Products; Fig. 4A). DAP22C is a peptidyl aminoalkanephosphonate that has been reported to target the serine proteases cathepsin G and chymotrypsin in vitro (25Oleksyszyn J. Powers J.C. Irreversible inhibition of serine proteases by peptide derivatives of (α-aminoalkyl)phosphonate diphenyl esters.Biochemistry. 1991; 30: 485-493Crossref PubMed Scopus (191) Google Scholar). However, it has not been used for any proteomics applications. We used MTS-I-49 to label mouse liver extracts and analyze the labeled sample by both SDS-PAGE (Fig. 4B) and by direct chemical cleavage or on-bead digestion (Fig. 4C). Although the on-bead digest again identified a large number of background and endogenously biotinylated proteins, the chemical cleavage revealed predominant recovery of the S9 family clan SC serine hydrolase, carboxylesterase 4. Overall the level of background peptides was decreased by over 90% when chemical elution was used for the sample preparation (Fig. 4C). Although the number of carboxylesterase peptides identified was reduced compared with the on-bead sample we could still make a confident identification with nearly 40% coverage of peptide sequence (Fig. 4D). Carboxylesterase 4 has not been characterized on a functional level and has never been confirmed to be catalytically active (26Rawlings N.D. Morton F.R. Barrett A.J. MEROPS: the peptidase database.Nucleic Acids Res. 2006; 34: D270-D272Crossref PubMed Scopus (464) Google Scholar). It is not clear why we were only able to identify this one esterase, but the lack of other reasonable targets in the on-bead digestion sample suggests that the probe may simply be inefficient or highly selective for targets that are not active in mouse liver extracts.Fig. 4Use of the cleavable linker probe for identification of serine hydrolases in mouse liver.A, structure of the phenoxy probe MTS-I-49 that contains the cleavable linker and is similar in structure to the commercially available serine protease probe DAP22C. B, mouse liver extracts were labeled with MTS-I-49 and analyzed by direct SDS-PAGE followed by biotin blotting. C, list of proteins identified by LC/MS/MS analysis of on bead-digested or chemically eluted proteins from mouse liver extracts labeled with MTS-I-49. A hypothetical protein was identified as the primary probe target after chemical cleavage. BLAST (Basic Local Alignment Search Tool) database searching identified it as liver carboxylesterase 4 (D). A number of additional endogenously biotinylated proteins were identified in the on bead-digested sample. The four most intensely labeled proteins from the gel image in B (labeled 1–4) likely represent the top four hits in the MS analysis in C based on predicted molecular weights. The proposed assignments are indicated using numbers corresponding to the number of the hits in the table in C. PhO, phosphonate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Finally, we were interested to see whether the active site peptides that are modified with the probe could be isolated and identified by MS/MS analysis. This is particularly important for applications of the cleavable linker system to ICAT where probe-labeled peptides are exclusively recovered whereas non-labeled peptides are washed away. Because the active site peptides of all of the cysteine cathepsins are difficult to identify due to their large size as trypsin-digested fragments, we tested the SV1 probe on purified bovine spleen cathepsin B. After on-bead digestion of enriched labeled cathepsins, active site peptides were eluted by chemical cleavage, and active site peptides were identified from the MS/MS fragmentation pattern. Surprisingly, not only cathepsin B but also cathepsin S was identified in this sample. The molecular mass of the precursor ion and its MS/MS fragmentation pattern showed the expected mass addition of 571 Da to the active site cysteine corresponding to the cleaved probe fragment (Fig. 5). These data confirm that the probe labeled active site cysteine residues and that it could be used to specifically isolate and sequence the active site peptides labeled in the target proteases. In all of our labeling and enrichment experiments, we never identified any peptides derived from the pro regions of the zymogen forms of any proteases (Fig. 6). These data fit with the finding that the probe only showed modification of proteins in the size rage of the mature cathepsins when the samples were analyzed by SDS-PAGE and confirm that our probe labels only catalytically active proteases.Fig. 5Detection of labeled active site peptides of cathepsins B and S. Purified bovine cathepsin B (Calbiochem) was labeled with SV1 (10 μm) and bound to immobilized streptavidin. The sample was digested on bead followed by washing to remove non-active site labeled peptides. The labeled active site peptides remained bound to the beads and were specifically eluted by reduction cleavage with sodium hydrosulfite. MS/MS analysis revealed the presence of active site peptides of both cathepsins B (A) and S (B) suggesting that the commercial sample was contaminated with cathepsin S. Due to the large molecular mass of the active site peptides, only triply charged species were detected. The tables at left show assignments of b and y ion series (detected ions are marked) in the MS/MS scans of precursor ions; the base peaks and MS/MS spectra are shown at right.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6Amino acid coverage of identified cathepsins. Positions of peptides identified in all labeling experiments (on bead-digested and chemically cleaved) are shown mapped onto the amino acid sequences of each target protease (lines below sequences). Cathepsin pro regions are shown in gray. None of the peptides mapped to the pro regions of any of the targets indicating that zymogen forms of the proteases were not labeled by the probes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In conclusion, our results using multiple activity-based probes that contain the chemically cleavable diazobenzene linker suggest that these probes will be valuable tools for direct proteomics profiling of active serine and cysteine proteases. The major advantage of this cleavable linker system is its ability to reduce background signals resulting in reduction in the number of both false positives and false negatives. We believe this linker system will have broad value for all types of small molecule proteomics probes that rely on affinity purification methods as it will help to reduce the time-consuming and expensive process of validating proteomics hits. In the examples presented here we had the benefit of significant knowledge of the types of targets we expected to identify with our probes. In ca" @default.
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